The initiation of an immune response against a specific antigen in mammals is brought about by the presentation of that antigen to T-cells. An antigen is presented to T-cells in the context of a major histocompatibility (MHC) complex. MHC complexes are located on the surface of antigen presenting cells (APCs); the 3-dimensional structure of MHCs includes a groove or cleft into which the presented antigen fits. When an appropriate receptor on a T-cell interacts with the MHC/antigen complex on an APC in the presence of necessary co-stimulatory signals, the T-cell is stimulated, triggering various aspects of the well characterized cascade of immune system activation events, including induction of cytotoxic T-cell function, induction of B-cell function and stimulation of cytokine production.
There are two basic classes of MHC molecules in mammals, MHC class I and MHC II. Both classes are large protein complexes formed by association of two separate proteins. Each class includes trans-membrane domains that anchor the complex into the cell membrane. MHC class I molecules are formed from two non-covalently associated proteins, the xcex1 chain and xcex22-microglobulin. The xcex1 chain comprises three distinct domains, xcex11, xcex12 and xcex13. The three dimensional structure of the xcex11 and xcex12 domains forms the groove into which antigens fit for presentation to T-cells. The xcex13 domain is a trans-membrane Ig-fold like domain that anchors the xcex1 chain into the cell membrane of the APC. MHC class I complexes, when associated with antigen (and in the presence of appropriate co-stimulatory signals) stimulate CD8 cytotoxic T-cells, which function to kill any cell which they specifically recognize.
The two proteins which associate non-covalently to form MHC class II molecules are termed the xcex1 and xcex2 chains. The xcex1 chain comprises xcex11 and xcex12 domains, and the xcex2 chain comprises xcex21 and xcex22 domains. The cleft into which the antigen fits is formed by the interaction of the xcex11 and xcex21 domains. The xcex12 and xcex22 domains are trans-membrane Ig-fold like domains that anchors the xcex1 and xcex2 chains into the cell membrane of the APC. MHC class II complexes, when associated with antigen (and in the presence of appropriate co-stimulatory signals) stimulate CD4 T-cells. The primary functions of CD4 T-cells are to initiate the inflammatory response and to regulate other cells in the immune system.
The genes encoding the various proteins that constitute the MHC complexes have been extensively studied in humans and other mammals. In humans, MHC molecules (with the exception of class I xcex22-microglobulin) are encoded in the HLA region, which is located on chromosome 6 and constitutes over 100 genes. There are 3 class I MHC xcex1 protein loci, termed HLA-A, -B and -C. There are also 3 pairs of class II MHC xcex1 and xcex2 chain loci, termed HLA-DR(A and B), HLA-DP(A and B), and HLA-DQ(A and B). In rats, the class I xcex1 gene is termed RT1.A, while the class II genes are termed RT1.Bxcex1 and RT1.Bxcex2. More detailed background information on the structure, function and genetics of MHC complexes can be found in Immunobiology: The Immune System in Health and Disease by Janeway and Travers, Cuurent Biology Ltd./Garland Publishing, Inc. (1997) (ISBN 0-8153-2818-4), and in Bodmer et al. (1994) xe2x80x9cNomenclature for factors of the HLA systemxe2x80x9d Tissue Antigens vol. 44, pages 1-18 (with periodic updates).
The key role that MHC complexes play in triggering immune recognition has led to the development of methods by which these complexes are used to modulate the immune response. For example, activated T-cells which recognize xe2x80x9cselfxe2x80x9d antigens (autoantigens) are known to play a key role in autoimmune diseases (such as rheumatoid arthritis and multiple sclerosis). Building on the observation that isolated MHC class II molecules (loaded with the appropriate antigen) can substitute for APCs carrying the MHC class II complex and can bind to antigen-specific T-cells, a number of researchers have proposed that isolated MHC/antigen complexes may be used to treat autoimmune disorders. Thus U.S. Pat. No. 5,194,425 (Sharma et al.) and U.S. Pat. No. 5,284,935 (Clark et al.) disclose the use of isolated MHC class II complexes loaded with a specified autoantigen and conjugated to a toxin to eliminate T-cells that are specifically immunoreactive with autoantigens. In another context, it has been shown that the interaction of isolated MHC II/antigen complexes with T-cells, in the absence of co-stimulatory factors, induces a state of non-responsiveness known as anergy. (Quill et al., J. Immunol., 138:3704-3712 (1987)). Following this observation, Sharma et al. (U.S. Pat. Nos. 5,468,481 and 5,130,297) and Clarke et al. (U.S. Pat. No. 5,260,422) have suggested that such isolated MHC II/antigen complexes may be administered therapeutically to anergize T-cell lines which specifically respond to particular autoantigenic peptides.
Methods for using isolated MHC complexes in the detection, quantification and purification of T-cells which recognize particular antigens have been studied for use in diagnostic and therapeutic applications. By way of example, early detection of T-cells specific for a particular autoantigen would facilitate the early selection of appropriate treatment regimes. The ability to purify antigen-specific T-cells would also be of great value in adoptive immunotherapy. Adoptive immunotherapy involves the removal of T-cells from a cancer patient, expansion of the T-cells in vitro and then reintroduction of the cells to the patient (see U.S. Pat. No. 4,690,915; Rosenberg et al. New Engl. J. Med. 319:1676-1680 (1988)). Isolation and expansion of cancer specific T-cells with inflammatory properties would increase the specificity and effectiveness of such an approach.
To date, however, attempts to detect, quantify or purify antigen specific T-cells using isolated MvHC/antigen complexes have not met with widespread success because, among other reasons, binding between the T-cells and such isolated complexes is transient and hence the T-cell/MHC/antigen complex is unstable. In an attempt to address these problems, Altman et al. (Science 274, 94-96 (1996) and U.S. Pat. No. 5,635,363) have proposed the use of large, covalently linked multimeric structures of MHC/antigen complexes to stabilize this interaction by simultaneously binding to multiple T-cell receptors on a target T-cell.
Although the concept of using isolated MHC/antigen complexes in therapeutic and diagnostic applications holds great promise, a major drawback to the various methods reported to date is that the complexes are large and consequently difficult to produce and to work with. While the complexes can be isolated from lymphocytes by detergent extraction, such procedures are inefficient and yield only small amounts of protein. The cloning of the genes encoding the various MHC complex subunits has facilitated the production of large quantities of the individual subunits through expression in prokaryotic cells, but the assembly of the individual subunits into MHC complexes having the appropriate conformational structure has proven difficult.
This invention is founded on the discovery that mammalian MHC function can be mimicked through the use of recombinant polypeptides that include only those domains of MHC molecules, that define the antigen binding cleft. These molecules are useful to detect, quantify and purify antigen-specific T-cells. The molecules provided herein may also be used in clinical and laboratory applications to detect, quantify and purify antigen-specific T-cells, induce anergy in T-cells, as well as to stimulate T-cells, and to treat diseases mediated by antigen-specific T-cells.
By way of example, while Altman et al. (U.S. Pat. No. 5,635,363) contemplate the use of multimers of MHC class II complexes comprising xcex11, xcex12, xcex21 and xcex22 domains and associated peptide antigens, to bind to and purify antigen-specific T-cells from a mixture, the present inventors have discovered that such antigen-specific T-cell binding can be accomplished with a much simpler monomeric molecule comprising, in the case of class II MHC molecules, only the xcex11 and xcex21 domains in covalent linkage (and in association with an antigenic determinant). For convenience, such MHC class II polypeptides are hereinafter referred to as xe2x80x9cxcex21xcex11xe2x80x9d. Equivalent molecules derived from MHC class I molecules are also provided by this invention. Such molecules comprise the xcex11 and xcex12 domains of class I molecules in covalent linkage and in association with an antigenic determinant. Such MHC class I polypeptides are referred to as xe2x80x9cxcex11xcex12xe2x80x9d. These two domain molecules may be readily produced by recombinant expression in prokaryotic or eukaryotic cells, and readily purified in large quantities. Moreover, these molecules may easily be loaded with any desired peptide antigen, making production of a repertoire of MHC molecules with different T-cell specificities a simple task.
It is shown that, despite lacking the trans-membrane Ig fold domains that are part of intact MHC molecule, these two domain MHC molecules refold in a manner that is structurally analogous to xe2x80x9cwholexe2x80x9d MHC molecules, and bind peptide antigens to form stable MHC/antigen complexes. Moreover, these two domain MHC/epitope complexes bind T-cells in an epitope-specific manner, and inhibit epitope-specific T-cell proliferation in vitro. In addition, administration of xcex21xcex11 molecules loaded with the myelin basic protein (MBP) epitope comprising amino acids 69-89 of MBP to rats is shown to both suppress the onset of and treat experimental autoimmune encephalomyelitis (EAE) in rats. Thus, the two domain MHC molecules display powerful and epitope-specific effects on T-cell activation both in vitro and in vivo. As a result, the disclosed MHC molecules are useful in a wide range of both in vivo and in vitro applications.
Various formulations of these two domain molecules are provided by the invention. In their most basic form, the two domain MHC class II molecules comprise xcex11 and xcex21 domains of a mammalian MHC class II molecule wherein the amino terminus of the xcex11 domain is covalently linked to the carboxy terminus of the xcex21 domain and wherein the polypeptide does not include the xcex12 or xcex22 domains. The two domain MHC class I molecules comprise an a xcex11 and xcex12 domains of a mammalian class I molecule, wherein the amino terminus of the xcex12 domain is covalently linked to the carboxy terminus of the xcex11 domain, and wherein the polypeptide does not include an MHC class I xcex13 domain. For most applications, these molecules are associated, by covalent or non-covalent interaction, with an antigenic determinant, such as a peptide antigen. In certain embodiments, the peptide antigen is covalently linked to the amino terminus of the xcex21 domain of the class II molecules, or the xcex11 domain of the class I molecules. The two domain molecules may also comprise a detectable marker, such as a fluorescent label or a toxic moiety, such as ricin A.
The invention also provides nucleic acid molecules that encode the two domain MHC molecules, as well as expression vectors that may be conveniently used to express these molecules. In particular embodiments, the nucleic acid molecules include sequences that encode the antigenic peptide as well as the two domain MHC molecule. For example, one such nucleic acid molecule may be represented by the formula Pr-P-B-A, wherein Pr is a promoter sequence operably linked to P (a sequence encoding the peptide antigen), B is the class I xcex11 or the class II xcex21 domain, and A is the class I xcex12 domain or the class II xcex11 domain. In these nucleic acid molecules, P, B and A comprise a single open reading frame, such that the peptide and the two MHC domains are expressed as a single polypeptide chain.
In vitro, the two domain MHC molecules may be used to detect and quantify T-cells, and regulate T-celt function. Thus, such molecules loaded with a selected antigen may be used to detect, monitor and quantify the population of a T-cells that are specific for that antigen. The ability to do this is beneficial in a number of clinical settings, such as monitoring the number of tumor antigen-specific T-cells in blood removed from a cancer patient, or the number of self-antigen specific T-cells in blood removed from a patient suffering from an autoimmune disease. In these contexts, the disclosed molecules are powerful tools for monitoring the progress of a particular therapy. In addition to monitoring and quantifying antigen-specific T-cells, the disclosed molecules may also be used to purify such cells for adoptive immunotherapy. Thus, the disclosed MHC molecules loaded with a tumor antigen may be used to purify tumor-antigen specific T-cells from a cancer patient. These cells may then be expanded in vitro before being returned to the patient as part of a cancer treatment. When conjugated with a toxic moiety, the two domain molecules may be used to kill T-cells having a particular antigen specificity. Alternatively, the molecules may also be used to induce anergy in such T-cells.
The two domain molecules may also be used in vivo to target specified antigen-specific T-cells. By way of example, a xcex21xcex11 molecule loaded with a portion of myelin basic protein (MBP) and administered to patients suffering from multiple sclerosis may be used to induce anergy in NIBP-specific T-cells, thus alleviating the disease symptoms. Alternatively, such molecules may be conjugated with a toxic moiety to more directly kill the disease-causing T-cells.
These and other aspects of the invention are described in more detail in the following sections.